Glenda Courtney-Martin

The In Vivo Sparing of Methionine by Cysteine in Sulfur Amino Acid Requirements in Animal Models and Adult Humans

Sulfur amino acid metabolism has been receiving increased attention because of the link to chronic diseases such as cardiovascular disease, Alzheimers disease, and diabetes. In addition, the role of cysteine and optimal intakes for physiological substrates such as glutathione are currently of considerable interest in human health. Although the dietary indispensability of methionine is not in question, the ability of cysteine to substitute for a portion of its requirement has been the topic of much debate. Methionine is often the most limiting amino acid in the diets of the developing worlds population because of its low concentration in cereal grains. Therefore, the ability of cysteine to substitute for methionine requirement is not just biologically interesting; it is also of considerable economic and social importance. The primary goal of this review is to discuss the available evidence on the effect of cysteine substitution for methionine to meet the total sulfur amino acid requirement in adult humans, including an assessment of the methodological features of experiments with conflicting results. Assessment of the requirement experiments for amino acids with complex metabolism such as methionine and cysteine must begin with a careful definition of requirements and what substitution means. As a result of these definitions, a set of criteria for the intakes of methionine that will allow demonstration of the substitution effect have been developed. Some recent publications are assessed using these definitions and criteria, and a possible reason for the conflicting results in the literature is proposed. An approach to estimating tolerable upper intakes is also proposed. Research on in vivo sulfur amino acid metabolism in humans is tremendously difficult, and therefore, we do not wish to be overly critical of the high-quality work of the ambitious and highly intelligent men and women who have conducted various studies. Our goal is to objectively review the data for the reader in a logical and comprehensive manner and propose methods that may avoid difficulties in future studies.

Dietary Protein Requirement of Female Adults >65 Years Determined by the Indicator Amino Acid Oxidation Technique Is Higher Than Current Recommendations

BACKGROUND Studies on protein requirements in vulnerable groups such as older adults are few, and results are conflicting. OBJECTIVE The main objective of this study was to determine the protein requirements of free-living women >65 y by measuring the oxidation of l-[1-(13)C]phenylalanine to (13)CO2 in response to graded intakes of protein. METHODS Twelve subjects participated in the study, with protein intakes ranging from 0.2 to 2.0 g · kg(-1) · d(-1) for a total of 82 studies. The diets provided energy at 1.5 times each subjects resting energy expenditure and were isocaloric. Protein was given as an amino acid mixture on the basis of the egg protein pattern, except for phenylalanine and tyrosine, which were maintained constant across the protein intake amounts. All subjects were adapted for 2 d before the study day to a protein intake of 1.0 g · kg(-1) · d(-1). The mean protein requirement was determined by applying a mixed-effects change-point regression analysis to F(13)CO2 (label tracer oxidation in (13)CO2 breath), which identified a breakpoint in the F(13)CO2 in response to graded amounts of protein. RESULTS The mean estimated average requirement (EAR) and upper 95% CI (approximating the RDA) protein requirement of women >65 y were 0.96 and 1.29 g · kg(-1) · d(-1), respectively. CONCLUSION These estimates of protein requirements for older women are higher than the current EAR and RDA based on nitrogen balance data, which are 0.66 and 0.80 g · kg(-1) · d(-1), respectively. This trial was registered at clinicaltrials.gov as NCT01604980.

Measurement of homocysteine and related metabolites in human plasma and urine by liquid chromatography electrospray tandem mass spectrometry

The sulfur amino acids, methionine and cysteine play crucial roles in cells as a substrate for protein synthesis, as a methyl donor, and for the synthesis of sulfur-containing compounds, including the key intracellular tripeptide, glutathione. Homocysteine is an intermediary metabolite formed during the metabolism of methionine to cysteine. Dysregulation of homocysteine metabolism is implicated in adverse clinical outcomes such as increased risk of cardiovascular disease, stroke, Alzheimers disease dementia and osteoporosis. While hyperhomocysteinemia is commonly observed in those conditions, the impact on other related metabolites is condition-specific. Therefore, there exists a need to establish precise and sensitive analytical techniques that allow for the simultaneous measurement of homocysteine and related metabolites in biological samples. The current review outlines the development and use of liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) to simultaneously measure metabolites involved in sulfur amino acid metabolism. Additionally, extensions of the technique in relation to the measurement of sulfur amino acid and one-carbon kinetics in vivo are discussed. The LC-MS/MS technique has the capacity for unambiguous analyte identification and confirmation, due to its high specificity and sensitivity. It has the greatest potential of being accepted and utilized as a dedicated homocysteine and its related metabolite Standard reference method (SRM).

Threonine requirement of parenterally fed postsurgical human neonates

BACKGROUND The threonine requirement of human neonates who receive parenteral nutrition (PN) has not been determined experimentally. OBJECTIVE The objective was to determine the parenteral threonine requirement for human neonates by using the minimally invasive indicator amino acid oxidation technique with L-[1-(13)C]phenylalanine as the indicator amino acid. DESIGN Nine postsurgical neonates were randomly assigned to 16 threonine intakes ranging from 10 to 100 mg . kg(-1) . d(-1). Breath and urine samples were collected at baseline and at plateau for (13)CO(2) and amino acid enrichment, respectively. The mean threonine requirement was determined by applying a 2-phase linear regression crossover analysis to the measured rates of (13)CO(2) release (F(13)CO(2)) and L-[1-(13)C]phenylalanine oxidation. RESULTS The mean threonine parenteral requirement determined by using phenylalanine oxidation was 37.6 mg . kg(-1) . d(-1) (upper and lower confidence limits, respectively: 29.9 and 45.2 mg . kg(-1) . d(-1)) and by using F(13)CO(2) oxidation was 32.8 mg . kg(-1) . d(-1) (upper and lower confidence limits, respectively: 29.7 and 35.9 mg . kg(-1) . d(-1)). Graded intakes of threonine had no effect on phenylalanine flux. CONCLUSION This is the first study to report on the threonine requirement for human neonates receiving PN. We found that the threonine requirement for postsurgical PN-fed neonates is 22-32% of the content of threonine that is presently found in commercial PN solutions (111-165 mg . kg(-1) . d(-1)).

Lysine requirement in parenterally fed postsurgical human neonates

BACKGROUND The lysine requirement of human neonates receiving parenteral nutrition (PN) has not been determined experimentally. OBJECTIVE The objective was to determine the parenteral lysine requirement for human neonates by using the minimally invasive indicator amino acid oxidation technique with l-[1-(13)C] phenylalanine as the indicator amino acid. DESIGN Eleven postsurgical neonates were randomly assigned to 15 lysine intakes ranging from 50 to 260 mg . kg(-1) . d(-1). Breath and urine samples were collected at baseline and at plateau for (13)CO(2) (F(13)CO(2)) and amino acid enrichment, respectively. The mean lysine requirement was determined by applying a 2-phase linear regression crossover analysis to the measured rates of F(13)CO(2) release and l-[1-(13)C]phenylalanine oxidation. RESULTS The mean parenteral lysine requirement determined by F(13)CO(2) release oxidation was 104.9 mg . kg(-1) . d(-1) (upper and lower CIs: 120.6 and 89.1 mg . kg(-1) . d(-1), respectively). The mean lysine parenteral requirement determined by phenylalanine oxidation was 117.6 mg . kg(-1) . d(-1) (upper and lower CIs: 157.5 and 77.6 mg . kg(-1) . d(-1), respectively). Graded intakes of lysine had no effect on phenylalanine flux. CONCLUSION We recommend a mean lysine requirement for the postsurgical PN-fed neonate of 104.9 mg . kg(-1) . d(-1), which is 32-43% of the lysine concentration presently found in commercial PN solutions (246-330 mg . kg(-1) . d(-1)). This trial was registered at clinicaltrials.gov as NCT00779753.

Methionine-Adequate Cysteine-Free Diet Does Not Limit Erythrocyte Glutathione Synthesis in Young Healthy Adult Men

Most methods of determining amino acid (AA) requirements are based on endpoints that determine adequacy for protein synthesis. However, the sulfur AA (SAA) cysteine is believed to be the rate-limiting substrate for synthesis of the most abundant intracellular antioxidant, glutathione (GSH). Our objectives were to determine whether supplementation of cysteine in a diet containing adequate SAA for protein synthesis, as methionine, increased GSH synthesis by measuring the fractional and absolute synthesis rates, and if concentration of GSH changed in response to feeding 5 graded intakes of cysteine (0, 10, 20, 30, and 40 mg x kg(-1) x d(-1)) in a random order with a fixed methionine intake of 14 mg x kg(-1) x d(-1) and a protein intake of 1 g x kg(-1) x d(-1). Each subject received a multivitamin and choline supplement during the study. Four healthy adult men each underwent 5 isotope infusion studies of 7-h duration after a 2-d adaptation to the level of cysteine intake being studied on the isotope infusion day. The isotope used was [U-(13)C(2)-(15)N]glycine. Analyses included erythrocyte GSH synthesis rates and concentration and urinary sulfate excretion. The GSH synthesis rates and concentration, measured at a methionine intake of 14 mg x kg(-1) x d(-1), did not change with increasing intakes of cysteine. Urinary sulfate excretion showed a significant positive relationship with cysteine intake (r = 0.92; P < 0.01). In conclusion, this study provides preliminary evidence that consumption of SAA adequate to meet the requirement for protein synthesis does not limit GSH synthesis in healthy adult men receiving an otherwise adequate diet.

The Addition of Cysteine to the Total Sulphur Amino Acid Requirement as Methionine Does Not Increase Erythrocytes Glutathione Synthesis in the Parenterally Fed Human Neonate

Controversy exists as to whether the parenterally (PN) fed human neonate is capable of synthesizing adequate cysteine from methionine if the total dietary requirement for sulfur amino acid (SAA) is provided as methionine only. The goal of this study was to gather data on whether glutathione (GSH) synthesis is maximized at a methionine intake previously shown to be adequate for protein synthesis in the PN-fed human neonate. We measured GSH concentration, fractional, and absolute synthesis rate in five PN-fed human neonates. Each neonate underwent two isotope infusion studies of 7 h duration after a 2-d adaptation to the total SAA requirement (methionine only) and again after a further 2-d adaptation to the same methionine intake supplemented with cysteine at 10 mg · kg−1 · d−1. Cysteine supplementation did not significantly affect GSH synthesis. These data suggest that term infants are capable of synthesizing cysteine from methionine, not only for protein but also for GSH synthesis.

Protein Requirements during Aging.

Protein recommendations for elderly, both men and women, are based on nitrogen balance studies. They are set at 0.66 and 0.8 g/kg/day as the estimated average requirement (EAR) and recommended dietary allowance (RDA), respectively, similar to young adults. This recommendation is based on single linear regression of available nitrogen balance data obtained at test protein intakes close to or below zero balance. Using the indicator amino acid oxidation (IAAO) method, we estimated the protein requirement in young adults and in both elderly men and women to be 0.9 and 1.2 g/kg/day as the EAR and RDA, respectively. This suggests that there is no difference in requirement on a gender basis or on a per kg body weight basis between younger and older adults. The requirement estimates however are ~40% higher than the current protein recommendations on a body weight basis. They are also 40% higher than our estimates in young men when calculated on the basis of fat free mass. Thus, current recommendations may need to be re-assessed. Potential rationale for this difference includes a decreased sensitivity to dietary amino acids and increased insulin resistance in the elderly compared with younger individuals.

Plasma Aluminum Concentrations in Pediatric Patients Receiving Long-Term Parenteral Nutrition

BACKGROUND Patients receiving long-term parenteral nutrition (PN) are at increased risk of aluminium (Al) toxicity because of bypass of the gastrointestinal tract during PN infusion. Complications of Al toxicity include metabolic bone disease (MBD), Al-associated encephalopathy in adults, and impaired neurological development in preterm infants. Unlike the United States, there are no regulations regarding Al content of large- and small-volume parenterals in Canada. We, therefore, aimed to present our data on plasma Al concentration and Al intake from our cohort of pediatric patients receiving long-term PN. METHODS Plasma Al concentration was retrospectively gathered from the patient charts of all 27 patients with intestinal failure (IF) receiving long-term PN at The Hospital for Sick Children, Toronto, Canada, and compared with age- and sex-matched controls recruited for comparison. In addition, Al concentration was measured in PN samples collected from 10 randomly selected patients with IF and used to determine their Al intake. RESULTS The plasma Al concentration of patients with IF receiving long-term PN was significantly higher than that of control participants (1195 ± 710 vs 142 ± 63 nmol/L; P < .0001). In the subgroup of 10 patients for whom Al intake from their PN solution was determined, mean ± SD Al intake from PN was 15.4 ± 15 µg/kg, 3 times the Food and Drug Administration upper recommended intake level, and Al intake was significantly related to plasma Al concentration (P = .02, r (2) = 0.52). CONCLUSION Pediatric patients receiving long-term PN for IF in Canada are at risk for Al toxicity.

Sulfur amino acid metabolism and requirements.

The sulfur amino acids (SAA) are methionine and cysteine. Methionine is indispensable.1–5 It donates its sulfur atom to cysteine during the process of trans-sulfuration, making cysteine a dispensable amino acid.6 The carbon skeleton of cysteine, however, is donated by serine.6 Methionine is metabolized via three major metabolic pathways: transmethylation, remethylation, and trans-sulfuration. It serves as the major methyl group donor in vivo,7,8 while cysteine is the rate-limiting substrate for the synthesis of the antioxidant glutathione (GSH).9–11 Both amino acids are important for protein synthesis. The total sulfur amino acid (TSAA) requirement, when fulfilled by the provision of methionine, can be significantly reduced only by adding cysteine to the diet. This phenomenon has been firmly established in animals12–15 and in humans16–18; it is consistent across the lifespan, having been observed in neonates,5–20 children,21 and adults.17,18 In the present issue of this journal, F. Jahoor examines the role the availability of sulfur amino acids plays in severe childhood undernutrition.22 The present editorial complements that work by providing an overview of SAA metabolism and requirements. ### Methionine In healthy adults, the metabolism of methionine is regulated toward anabolism23 in the fed state, and methionine flux is increased relative to that observed in the fasted state. Feeding results in decreased methionine release from protein breakdown and increased transmethylation, trans-sulfuration, and remethylation. In the fasted state, methionine utilization for protein synthesis is increased relative to transmethylation, resulting in the conservation of methionine when SAA availability is low. When SAAs are absent from the diet of healthy adults,17 methionine is used preferentially for protein synthesis relative to transmethylation, confirming previous findings that methionine is conserved via protein synthesis when SAA …